【摘要】：惡性腫瘤是目前造成人類(lèi)死亡的三大殺手之一,嚴(yán)重威脅人類(lèi)的身體健康。而肝細(xì)胞癌是世界上最常見(jiàn)的腫瘤之一,每年肝癌導(dǎo)致全球662000人死亡,其中大約一半患者出現(xiàn)在中國(guó)。另外,肝癌治療還具有預(yù)后不良、易復(fù)發(fā)和易轉(zhuǎn)移等特點(diǎn)。目前,肝癌的治療手段主要有化學(xué)治療、放射治療、手術(shù)治療、基因治療、經(jīng)皮治療和肝移植等六種方法,但這些手段不僅費(fèi)用高昂還容易引起極大的毒副作用。小牛脾提取物注射液作為多組分生化提取物具有多靶點(diǎn)、低毒性的特點(diǎn),但目前還沒(méi)有系統(tǒng)的研究闡述其抗腫瘤活性及相關(guān)機(jī)制。本文主要研究小牛脾提取物注射液的抗肝癌活性及其相關(guān)機(jī)制,并對(duì)其免疫調(diào)節(jié)作用進(jìn)行評(píng)價(jià)。本研究主要包括以下幾個(gè)方面:1.小牛脾提取物注射液體外抗肝癌活性及其機(jī)制的研究考察小牛脾提取物注射液(CSEI)對(duì)多種癌癥細(xì)胞的抑制率,其中,以肝癌細(xì)胞系Hep G2和SMMC-7721為主要研究對(duì)象考察小牛脾提取注射液對(duì)肝癌細(xì)胞系的促凋亡作用。同時(shí),采用生化分析手段考察注射液促肝癌細(xì)胞凋亡的機(jī)制。實(shí)驗(yàn)結(jié)果顯示,小牛脾提取物注射液在體外可以抑制人肝癌、乳腺癌和肺癌等細(xì)胞系的生長(zhǎng),其中注射液對(duì)肝癌細(xì)胞系SMMC-7721和Hep G2的抑制效果最明顯,而且注射液可以增加肝癌細(xì)胞中ROS的產(chǎn)生、改變線粒體膜電位、激活caspase級(jí)聯(lián)反應(yīng)等。此外,采用Western blot檢測(cè)細(xì)胞和腫瘤中凋亡相關(guān)蛋白PARP、cleaved-PARP、Bcl2、P38、phospho-P38、JNK、phospho-JNK1、ERK1/2、phospho-ERK1/2、cleaved-caspase3、cleaved-caspase 8和cleaved-caspase 9等的表達(dá)情況,同時(shí)顯著降低氧化應(yīng)激相關(guān)蛋白Nrf2和Catalase在肝癌細(xì)胞和腫瘤中的表達(dá)。綜上所述,小牛脾提取物注射液抑制癌細(xì)胞增殖并通過(guò)ROS相關(guān)的線粒體途徑誘導(dǎo)肝癌細(xì)胞的凋亡。2.小牛脾提取物注射液體內(nèi)抗肝癌活性及其機(jī)制的研究為了進(jìn)一步確定小牛脾提取物注射液的抗肝癌作用,建立Hep G2肝癌異位裸鼠模型。小牛脾提取物注射液治療14天內(nèi)考察整個(gè)治療期間小鼠體重和腫瘤體積。結(jié)束治療后,取腫瘤進(jìn)行Western blot實(shí)驗(yàn)分析抗腫瘤及氧化應(yīng)激相關(guān)蛋白質(zhì)表達(dá)情況。實(shí)驗(yàn)結(jié)果證明,小牛脾提取物注射液能夠使Hep G2肝癌異位模型中裸鼠的腫瘤體積顯著下降,并對(duì)小鼠體重沒(méi)有影響。與此同時(shí),Western blot結(jié)果與體內(nèi)實(shí)驗(yàn)一致。以上結(jié)果說(shuō)明小牛脾提取物注射液具有良好的抗肝癌活性能,其通過(guò)ROS/MAPKs依賴(lài)的線粒體途徑誘導(dǎo)肝癌細(xì)胞凋亡。3.小牛脾提取物注射液免疫調(diào)節(jié)活性的評(píng)價(jià)本文通過(guò)建立由環(huán)磷酰胺誘導(dǎo)的免疫低下模型研究小牛脾提取物注射液的免疫調(diào)節(jié)活性。經(jīng)過(guò)14天的治療,小牛脾提取物注射液(在臨床使用劑量范圍內(nèi))可以逆轉(zhuǎn)由環(huán)磷酰胺誘導(dǎo)的免疫力低下,顯著增加動(dòng)物脾臟指數(shù)并恢復(fù)血清中IL-2,6,10,12和IFN-α,γ以及TNF-α等免疫和炎癥相關(guān)細(xì)胞因子的水平,提高NK細(xì)胞殺傷活力和T淋巴的細(xì)胞的轉(zhuǎn)化能力,同時(shí)通過(guò)增加動(dòng)物脾臟中IKKβ的磷酸化作用于NFκB通路來(lái)調(diào)節(jié)動(dòng)物機(jī)體內(nèi)免疫和炎癥水平。綜上所述,CSEI是一種安全有效的抗肝癌藥物,同時(shí)具有調(diào)節(jié)機(jī)體免疫力的活性,其通過(guò)ROS/MAPKs依賴(lài)的線粒體通路誘導(dǎo)人肝癌細(xì)胞凋亡。本研究為小牛脾提取物注射液作為肝細(xì)胞癌和其他原發(fā)腫瘤的潛在治療劑提供藥理學(xué)證據(jù)。
[Abstract]:Malignant tumor is one of the three major killers that currently cause human death, which is a serious threat to human health. Hepatocellular carcinoma is one of the world's most common tumors, leading to a global 662,000 deaths each year, of which about half of the patients are in China. in addition, that treatment of the liver cancer has the characteristics of poor prognosis, easy recurrence and easy transfer, and the like. At present, the treatment of liver cancer mainly includes six methods such as chemotherapy, radiation therapy, surgical treatment, gene therapy, skin treatment and liver transplantation. As a multi-component biochemical extract, the extract of calf-spleen extract has the characteristics of multi-target and low toxicity, but there is no systematic study on its anti-tumor activity and related mechanism. In this paper, the anti-liver cancer activity and its related mechanism of the extract of calf-spleen extract were studied, and the immunoregulation effect was evaluated. The study mainly includes the following aspects: 1. The inhibitory rate of calf-spleen extract injection (CSEI) on various cancer cells was studied in vitro. The effect of calf-spleen extraction injection on the apoptosis of liver cancer cell line was studied with the liver cancer cell line Hep G2 and SMMC-7721 as the main research object. At the same time, the mechanism of the apoptosis of the liver cancer cells was examined by means of biochemical analysis. The experimental results show that the extract of calf spleen can inhibit the growth of cell lines such as human liver cancer, breast cancer and lung cancer in vitro, and the effect of injection on the SMMC-7721 and Hep G2 of the liver cancer cell line is most obvious, and the injection can increase the generation of ROS in the liver cancer cell, the mitochondrial membrane potential is changed, the caspase cascade reaction is activated, and the like. In addition, Western blot was used to detect the expression of apoptosis-related proteins PARP, clear-PARP, Bcl2, P38, phospho-P38, JNK, phospho-JNK1, ERK1/ 2, phospho-ERK1/ 2, clear-caspase3, clear-caspase-8, and clear-caspase-9 in the cells and tumors. In conclusion, calf spleen extract injection inhibits the proliferation of cancer cells and induces apoptosis of the liver cancer cells through the ROS-related mitochondrial pathway. The anti-liver cancer activity and its mechanism in the extract of calf-spleen extract were studied in order to further determine the anti-hepatoma effect of the extract of calf-spleen extract. The body weight and tumor volume of the mice during the course of the treatment were examined within 14 days of treatment with the calf spleen extract injection. After the treatment, the expression of anti-tumor and oxidative stress-related proteins was analyzed by Western blot. The results of the experiment show that the tumor volume of the nude mice in the Hep G2 liver cancer heterotopic model can be significantly reduced, and the weight of the mice is not affected. At the same time, the results of Western blot were consistent with in vivo experiments. The above results show that the extract of calf spleen extract has good anti-liver cancer activity, and it can induce the apoptosis of liver cancer cells through the mitochondrial pathway dependent on ROS/ MAPKs. The immunomodulatory activity of the extract of calf-spleen extract was evaluated by establishing an immune-low model induced by cyclophosphamide. After 14 days of treatment, the calf-spleen extract injection (in the clinical use dose range) can reverse the hypoimmunity induced by the cyclophosphamide, significantly increase the animal spleen index and restore the IL-2, 6, 10, 12 and IFN-1 in the serum, The levels of immune and inflammatory-related cytokines, such as TNF-1, and the ability of NK cell killing and T-Lymphatic cells are improved, and the level of immunity and inflammation in the body of an animal is regulated by increasing the phosphorylation of IKK in the spleen of the animal in the NF-B pathway. In conclusion, CSEI is a safe and effective anti-liver cancer drug, and has the activity of regulating the immunity of the body, and the mitochondrial pathway, which is dependent on the ROS/ MAPKs, induces the apoptosis of human liver cancer cells. The present study provides pharmacological evidence for calf spleen extract injection as a potential therapeutic agent for hepatocellular carcinoma and other primary tumors.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.7

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